ABSTRACT
The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine's binding orientations that may constitute an integral component of this SSRI's high affinity for hSERT. Herein, we investigate factors contributing to paroxetine's high affinity by modifying both the ligand and the protein. We generated a series of bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically similar but more electron-rich Br atom (13) had the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both wild type (WT) and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.
Subject(s)
Bromine/metabolism , Paroxetine/analogs & derivatives , Paroxetine/metabolism , Serotonin Plasma Membrane Transport Proteins/metabolism , Binding Sites/drug effects , Binding Sites/physiology , Bromine/chemistry , Crystallography, X-Ray/methods , HEK293 Cells , HeLa Cells , Humans , Protein Binding/drug effects , Protein Binding/physiology , Selective Serotonin Reuptake Inhibitors/metabolism , Selective Serotonin Reuptake Inhibitors/pharmacologyABSTRACT
The serotonin transporter (SERT) is one of the primary targets for medications to treat neuropsychiatric disorders and functions by exploiting pre-existing ion gradients of Na+, Cl-, and K+ to translocate serotonin from the synaptic cleft into the presynaptic neuron. Although recent hSERT crystal structures represent a milestone for structure-function analyses of mammalian neurotransmitter:sodium symporters, they are all derived from thermostabilized but transport-deficient constructs. Two of these structures are in complex with paroxetine, the most potent selective serotonin reuptake inhibitor known. In this study, by carrying out and analyzing the results of extensive and comparative molecular dynamics simulations while also re-evaluating the transport and binding properties of the thermostabilized constructs, we identified functionally important structural elements that are perturbed by these mutations, revealed unexpected dynamics in the central primary binding site of SERT, and uncovered a conceivable ambiguity in paroxetine's binding orientation. We propose that the favored entropy contribution plays a significant role in paroxetine's extraordinarily high affinity for SERT. Our findings lay the foundation for future mechanistic studies and rational design of high-affinity SERT inhibitors. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Subject(s)
Paroxetine/metabolism , Selective Serotonin Reuptake Inhibitors/metabolism , Serotonin Plasma Membrane Transport Proteins/metabolism , Biological Transport, Active , Entropy , Humans , Kinetics , Models, Molecular , Molecular Dynamics Simulation , Mutation/genetics , Protein Binding , Protein Conformation , Serotonin Plasma Membrane Transport Proteins/chemistryABSTRACT
Neurotransmitter:sodium symporters (NSSs) are integral membrane proteins responsible for the sodium-dependent reuptake of small-molecule neurotransmitters from the synaptic cleft. The symporters for the biogenic amines serotonin (SERT), dopamine (DAT), and norepinephrine (NET) are targets of multiple psychoactive agents, and their dysfunction has been implicated in numerous neuropsychiatric ailments. LeuT, a thermostable eubacterial NSS homolog, has been exploited as a model protein for NSS members to canvass the conformational mechanism of transport with a combination of X-ray crystallography, cysteine accessibility, and solution spectroscopy. Despite yielding remarkable insights, these studies have primarily been conducted with protein in the detergent-solubilized state rather than embedded in a membrane mimic. In addition, solution spectroscopy has required site-specific labeling of nonnative cysteines, a labor-intensive process occasionally resulting in diminished transport and/or binding activity. Here, we overcome these limitations by reconstituting unlabeled LeuT in phospholipid bilayer nanodiscs, subjecting them to hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), and facilitating interpretation of the data with molecular dynamics simulations. The data point to changes of accessibility and dynamics of structural elements previously implicated in the transport mechanism, in particular transmembrane helices (TMs) 1a and 7 as well as extracellular loops (ELs) 2 and 4. The results therefore illuminate the value of this strategy for interrogating the conformational mechanism of the more clinically significant mammalian membrane proteins including SERT and DAT, neither of which tolerates complete removal of endogenous cysteines, and whose activity is heavily influenced by neighboring lipids.
Subject(s)
Dopamine/chemistry , Neurotransmitter Agents/chemistry , Serotonin/chemistry , Sodium-Phosphate Cotransporter Proteins/chemistry , Biogenic Amines/chemistry , Biogenic Amines/metabolism , Crystallography, X-Ray , Cysteine/chemistry , Dopamine/metabolism , Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Molecular Dynamics Simulation , Neurotransmitter Agents/metabolism , Norepinephrine/chemistry , Norepinephrine/metabolism , Serotonin/metabolism , Sodium-Phosphate Cotransporter Proteins/metabolismABSTRACT
The serotonin transporter (SERT) is an integral membrane protein that exploits preexisting sodium-, chloride-, and potassium ion gradients to catalyze the thermodynamically unfavorable movement of synaptic serotonin into the presynaptic neuron. SERT has garnered significant clinical attention partly because it is the target of multiple psychoactive agents, including the antidepressant paroxetine (Paxil), the most potent selective serotonin reuptake inhibitor known. However, the binding site and orientation of paroxetine in SERT remain controversial. To provide molecular insight, we constructed SERT homology models based on the Drosophila melanogaster dopamine transporter and docked paroxetine to these models. We tested the predicted binding configurations with a combination of radioligand binding and flux assays on wild-type and mutant SERTs. Our data suggest that the orientation of paroxetine, specifically its fluorophenyl ring, in SERT's substrate binding site directly depends on this pocket's charge distribution, and thereby provide an avenue toward understanding and enhancing high-affinity antidepressant activity.
Subject(s)
Paroxetine/pharmacology , Selective Serotonin Reuptake Inhibitors/pharmacology , Serotonin Plasma Membrane Transport Proteins/drug effects , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chickens , Cocaine/metabolism , Dopamine Plasma Membrane Transport Proteins/chemistry , Dopamine Plasma Membrane Transport Proteins/genetics , Dopamine Plasma Membrane Transport Proteins/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Paroxetine/chemistry , Protein Conformation , Radioligand Assay , Sequence Alignment , Sequence Homology, Amino Acid , Serotonin/metabolism , Serotonin Plasma Membrane Transport Proteins/genetics , Selective Serotonin Reuptake Inhibitors/chemistryABSTRACT
Structural characterization of integral membrane proteins (MPs) demands that the samples be pure, monodisperse, and stable. Detergents are required to extract MPs from the lipid bilayer in which they reside and to stabilize them for downstream biophysical analyses. Some of the best MP-stabilizing detergents pose problems for cryo-EM studies, but in this issue of Structure, Hauer et al. (2015) now offer a solution called GraDeR.
Subject(s)
Cryoelectron Microscopy/methods , Membrane Proteins/chemistry , Membrane Proteins/isolation & purification , AnimalsABSTRACT
Ion-coupled secondary transport is utilized by multiple integral membrane proteins as a means of achieving the thermodynamically unfavorable translocation of solute molecules across the lipid bilayer. The chemical nature of these molecules is diverse and includes sugars, amino acids, neurotransmitters, and other ions. LeuT is a sodium-coupled, nonpolar amino acid symporter and eubacterial member of the solute carrier 6 (SLC6) family of Na(+)/Cl(-)-dependent neurotransmitter transporters. Eukaryotic counterparts encompass the clinically and pharmacologically significant transporters for γ-aminobutyric acid (GABA), glycine, serotonin (5-hydroxytryptamine, 5-HT), dopamine (DA), and norepinephrine (NE). Since the crystal structure of LeuT was first solved in 2005, subsequent crystallographic, binding, flux, and spectroscopic studies, complemented with homology modeling and molecular dynamic simulations, have allowed this protein to emerge as a remarkable mechanistic paradigm for both the SLC6 class as well as several other sequence-unrelated SLCs whose members possess astonishingly similar architectures. Despite yielding groundbreaking conceptual advances, this vast treasure trove of data has also been the source of contentious hypotheses. This chapter will present a historical scientific overview of SLC6s; recount how the initial and subsequent LeuT structures were solved, describing the insights they each provided; detail the accompanying functional techniques, emphasizing how they either supported or refuted the static crystallographic data; and assemble these individual findings into a mechanism of transport and inhibition.
Subject(s)
Bacteria/chemistry , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/genetics , Bacteria/metabolism , Bacterial Proteins/metabolism , Computational Biology/methods , Crystallization/methods , Electron Spin Resonance Spectroscopy/methods , Fluorescence Resonance Energy Transfer/methods , Models, Molecular , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Protein ConformationABSTRACT
Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H(+), like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H(+)-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.
Subject(s)
Bacterial Proteins/chemistry , Biogenic Monoamines/chemistry , Brevibacillus/chemistry , Carrier Proteins/chemistry , Vesicular Monoamine Transport Proteins/chemistry , Amino Acid Sequence , Animals , Arginine/chemistry , Arginine/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biogenic Monoamines/metabolism , Brevibacillus/genetics , Brevibacillus/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Drug Resistance, Bacterial , Escherichia coli/genetics , Escherichia coli/metabolism , Evolution, Molecular , Gene Expression , Histidine/chemistry , Histidine/metabolism , Models, Molecular , Molecular Sequence Data , Protein Folding , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Structural Homology, Protein , Structure-Activity Relationship , Substrate Specificity , Synaptic Transmission/physiology , Vesicular Monoamine Transport Proteins/genetics , Vesicular Monoamine Transport Proteins/metabolismABSTRACT
Uptake of neurotransmitters by sodium-coupled monoamine transporters of the NSS family is required for termination of synaptic transmission. Transport is tightly regulated by protein-protein interactions involving the small cytoplasmic segments at the amino- and carboxy-terminal ends of the transporter. Although structures of homologues provide information about the transmembrane regions of these transporters, the structural arrangement of the terminal domains remains largely unknown. Here, we combined molecular modeling, biochemical, and biophysical approaches in an iterative manner to investigate the structure of the 82-residue N-terminal and 30-residue C-terminal domains of human serotonin transporter (SERT). Several secondary structures were predicted in these domains, and structural models were built using the Rosetta fragment-based methodology. One-dimensional (1)H nuclear magnetic resonance and circular dichroism spectroscopy supported the presence of helical elements in the isolated SERT N-terminal domain. Moreover, introducing helix-breaking residues within those elements altered the fluorescence resonance energy transfer signal between terminal cyan fluorescent protein and yellow fluorescent protein tags attached to full-length SERT, consistent with the notion that the fold of the terminal domains is relatively well-defined. Full-length models of SERT that are consistent with these and published experimental data were generated. The resultant models predict confined loci for the terminal domains and predict that they move apart during the transport-related conformational cycle, as predicted by structures of homologues and by the "rocking bundle" hypothesis, which is consistent with spectroscopic measurements. The models also suggest the nature of binding to regulatory interaction partners. This study provides a structural context for functional and regulatory mechanisms involving SERT terminal domains.
Subject(s)
Models, Molecular , Serotonin Plasma Membrane Transport Proteins/chemistry , Serotonin Plasma Membrane Transport Proteins/metabolism , Amino Acid Sequence , Circular Dichroism , Cytoplasm/chemistry , Fluorescence Resonance Energy Transfer , Humans , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Serotonin Plasma Membrane Transport Proteins/geneticsABSTRACT
The scintillation proximity assay is a powerful technique for measuring radioligand binding to membrane transporters and has become an integral part of high-throughput drug discovery screening efforts. Here we adapt the method for use with purified LeuT, a prokaryotic secondary transporter, reconstituted into phospholipid bilayer nanodiscs. This application surmounts potential challenges with background interference from endogenously expressed proteins, aggregation and loss of binding activity often accompanying detergent solubilization from native cell membranes, and heterogeneity in size and transporter orientation, where at least some ligand binding sites are inaccessible, associated with reconstitution into lipid vesicles.
Subject(s)
Amino Acid Transport Systems, Neutral/analysis , Amino Acid Transport Systems, Neutral/metabolism , Bacterial Proteins/analysis , Bacterial Proteins/metabolism , Scintillation Counting , Ligands , Lipid Bilayers/metabolism , Nanostructures/chemistryABSTRACT
Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.
Subject(s)
Receptors, AMPA/chemistry , Receptors, AMPA/metabolism , Amino Acid Sequence , Amino Acids , Animals , Binding Sites , Cell Line , Conserved Sequence , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , Protein Multimerization , Protein Stability , Protein Structure, Tertiary , Protein Subunits/chemistry , Rats , SolutionsABSTRACT
Secondary transporters are workhorses of cellular membranes, catalyzing the movement of small molecules and ions across the bilayer and coupling substrate passage to ion gradients. However, the conformational changes that accompany substrate transport, the mechanism by which a substrate moves through the transporter, and principles of competitive inhibition remain unclear. We used crystallographic and functional studies on the leucine transporter (LeuT), a model for neurotransmitter sodium symporters, to show that various amino acid substrates induce the same occluded conformational state and that a competitive inhibitor, tryptophan (Trp), traps LeuT in an open-to-out conformation. In the Trp complex, the extracellular gate residues arginine 30 and aspartic acid 404 define a second weak binding site for substrates or inhibitors as they permeate from the extracellular solution to the primary substrate site, which demonstrates how residues that participate in gating also mediate permeation.
Subject(s)
Amino Acid Transport Systems/chemistry , Amino Acid Transport Systems/metabolism , Bacterial Proteins/chemistry , Leucine/metabolism , Symporters/chemistry , Symporters/metabolism , Tryptophan/pharmacology , Amino Acid Transport Systems/antagonists & inhibitors , Amino Acids/metabolism , Amino Acids/pharmacology , Bacterial Proteins/metabolism , Binding Sites , Binding, Competitive , Biological Transport , Crystallization , Crystallography, X-Ray , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Kinetics , Ligands , Models, Biological , Models, Molecular , Protein Conformation , Protein Structure, Tertiary , Sodium/metabolism , Symporters/antagonists & inhibitors , Tryptophan/metabolismABSTRACT
Ion-coupled secondary transport is utilized by a broad range of integral membrane proteins to catalyze the energetically unfavorable movement of solute molecules across a lipid bilayer. Members of the solute carrier 6 (SLC6) family, present in both prokaryotes and eukaryotes, are sodium-coupled symporters that play crucial roles in processes as diverse as nutrient uptake and neurotransmitter clearance. The crystal structure of LeuT, a bacterial member of this family, provided the first atomic-level glimpse into overall architecture, pinpointed the substrate and sodium binding sites and implicated candidate helices and residues in the "gating" conformational changes that accompany ion binding and release. The structure is consistent with a wealth of elegant biochemical data on the eukaryotic counterparts and has for the first time permitted the construction of accurate homology models that can be directly tested experimentally. Sequence identity is especially high near the substrate and sodium binding sites and, thus, molecular insights within these regions have been substantial. However, there are several topics relevant to transport mechanism, inhibition and regulation that structure/function studies of LeuT cannot adequately address, suggesting the need for a eukaryotic transporter crystal structure.
Subject(s)
Eukaryota/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Bacterial Proteins/chemistry , Humans , Protein ConformationABSTRACT
Sodium-coupled transporters are ubiquitous pumps that harness pre-existing sodium gradients to catalyse the thermodynamically unfavourable uptake of essential nutrients, neurotransmitters and inorganic ions across the lipid bilayer. Dysfunction of these integral membrane proteins has been implicated in glucose/galactose malabsorption, congenital hypothyroidism, Bartter's syndrome, epilepsy, depression, autism and obsessive-compulsive disorder. Sodium-coupled transporters are blocked by a number of therapeutically important compounds, including diuretics, anticonvulsants and antidepressants, many of which have also become indispensable tools in biochemical experiments designed to probe antagonist binding sites and to elucidate transport mechanisms. Steady-state kinetic data have revealed that both competitive and noncompetitive modes of inhibition exist. Antagonist dissociation experiments on the serotonin transporter (SERT) have also unveiled the existence of a low-affinity allosteric site that slows the dissociation of inhibitors from a separate high-affinity site. Despite these strides, atomic-level insights into inhibitor action have remained elusive. Here we screen a panel of molecules for their ability to inhibit LeuT, a prokaryotic homologue of mammalian neurotransmitter sodium symporters, and show that the tricyclic antidepressant (TCA) clomipramine noncompetitively inhibits substrate uptake. Cocrystal structures show that clomipramine, along with two other TCAs, binds in an extracellular-facing vestibule about 11 A above the substrate and two sodium ions, apparently stabilizing the extracellular gate in a closed conformation. Off-rate assays establish that clomipramine reduces the rate at which leucine dissociates from LeuT and reinforce our contention that this TCA inhibits LeuT by slowing substrate release. Our results represent a molecular view into noncompetitive inhibition of a sodium-coupled transporter and define principles for the rational design of new inhibitors.
Subject(s)
Antidepressive Agents, Tricyclic/metabolism , Bacteria/chemistry , Clomipramine/metabolism , Plasma Membrane Neurotransmitter Transport Proteins/chemistry , Plasma Membrane Neurotransmitter Transport Proteins/metabolism , Animals , Antidepressive Agents, Tricyclic/pharmacology , Binding Sites , Clomipramine/pharmacology , Crystallography, X-Ray , Drug Design , Kinetics , Models, Molecular , Plasma Membrane Neurotransmitter Transport Proteins/antagonists & inhibitors , Protein Binding , Protein Conformation , Sequence Homology , Sodium/metabolism , Sodium/pharmacologyABSTRACT
Excitatory neurotransmission mediated by NMDA (N-methyl-D-aspartate) receptors is fundamental to the physiology of the mammalian central nervous system. These receptors are heteromeric ion channels that for activation require binding of glycine and glutamate to the NR1 and NR2 subunits, respectively. NMDA receptor function is characterized by slow channel opening and deactivation, and the resulting influx of cations initiates signal transduction cascades that are crucial to higher functions including learning and memory. Here we report crystal structures of the ligand-binding core of NR2A with glutamate and that of the NR1-NR2A heterodimer with glutamate and glycine. The NR2A-glutamate complex defines the determinants of glutamate and NMDA recognition, and the NR1-NR2A heterodimer suggests a mechanism for ligand-induced ion channel opening. Analysis of the heterodimer interface, together with biochemical and electrophysiological experiments, confirms that the NR1-NR2A heterodimer is the functional unit in tetrameric NMDA receptors and that tyrosine 535 of NR1, located in the subunit interface, modulates the rate of ion channel deactivation.
Subject(s)
Receptors, N-Methyl-D-Aspartate/chemistry , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Binding Sites , Dimerization , Disulfides/chemistry , Disulfides/metabolism , Electrophysiology , Glutamic Acid/metabolism , Ion Channel Gating , Ligands , Models, Molecular , Oocytes , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Rats , Receptors, N-Methyl-D-Aspartate/genetics , Substrate SpecificityABSTRACT
Na+/Cl--dependent transporters terminate synaptic transmission by using electrochemical gradients to drive the uptake of neurotransmitters, including the biogenic amines, from the synapse to the cytoplasm of neurons and glia. These transporters are the targets of therapeutic and illicit compounds, and their dysfunction has been implicated in multiple diseases of the nervous system. Here we present the crystal structure of a bacterial homologue of these transporters from Aquifex aeolicus, in complex with its substrate, leucine, and two sodium ions. The protein core consists of the first ten of twelve transmembrane segments, with segments 1-5 related to 6-10 by a pseudo-two-fold axis in the membrane plane. Leucine and the sodium ions are bound within the protein core, halfway across the membrane bilayer, in an occluded site devoid of water. The leucine and ion binding sites are defined by partially unwound transmembrane helices, with main-chain atoms and helix dipoles having key roles in substrate and ion binding. The structure reveals the architecture of this important class of transporter, illuminates the determinants of substrate binding and ion selectivity, and defines the external and internal gates.
Subject(s)
Bacterial Proteins/chemistry , Chlorides/metabolism , Leucine/metabolism , Membrane Transport Proteins/chemistry , Neurotransmitter Agents/metabolism , Sodium/metabolism , Amino Acid Sequence , Bacteria/chemistry , Bacterial Proteins/metabolism , Binding Sites , Biological Transport , Crystallography, X-Ray , Hydrophobic and Hydrophilic Interactions , Membrane Transport Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Sequence Alignment , Structure-Activity Relationship , Water/metabolismABSTRACT
In Escherichia coli, the homodimeric Krebs cycle enzyme isocitrate dehydrogenase (EcIDH) is regulated by reversible phosphorylation of a sequestered active site serine. The phosphorylation cycle is catalyzed by a bifunctional protein, IDH kinase/phosphatase (IDH-K/P). To better understand the nature of the interaction between EcIDH and IDH-K/P, we have examined the ability of an IDH homologue from Bacillus subtilis (BsIDH) to serve as a substrate for the kinase and phosphatase activities. BsIDH exhibits extensive sequence and structural similarities with EcIDH, particularly around the phosphorylated serine. Our previous crystallographic analysis revealed that the active site architecture of these two proteins is almost completely conserved. We now expand the comparison to include a number of biochemical properties. Both IDHs display nearly equivalent steady-state kinetic parameters for the dehydrogenase reaction. Both proteins are also phosphorylated by IDH-K/P in the same ratio (1 mole of phosphate per mole of monomer), and this stoichiometric phosphorylation correlates with an equivalent inhibition of IDH activity. Furthermore, tandem electrospray mass spectrometry demonstrates that BsIDH, like EcIDH, is phosphorylated on the corresponding active site serine residue (Ser-104). Despite the high degree of sequence, functional, and structural congruence between these two proteins, BsIDH is surprisingly a much poorer substrate of IDH-K/P than is EcIDH, with Michaelis constants for the kinase and phosphatase activities elevated by 60- and 3,450-fold, respectively. These drastically disparate values might result from restricted access to the active site cavity and/or from the lack of a potential docking site for IDH-K/P.
Subject(s)
Bacillus subtilis/enzymology , Escherichia coli/enzymology , Isocitrate Dehydrogenase/chemistry , Isocitrate Dehydrogenase/physiology , Phosphoprotein Phosphatases/chemistry , Protein Serine-Threonine Kinases/chemistry , Binding Sites , Catalysis , Crystallography, X-Ray , Dimerization , Kinetics , Mass Spectrometry , Models, Molecular , Peptides/chemistry , Phosphorylation , Plasmids/metabolism , Protein Binding , Serine/chemistry , Substrate Specificity , Time FactorsABSTRACT
Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [(32)P]3a or of [(32)P]3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.